The overall objective of this proposal is to define the sequence of molecular events that regulate the initiation of SV40 DNA replication. This information is imporant in order to understand the function of an important tumor virus transforming protein and may ultimately be useful in identifying components of host cell replicons as well. We have the following specific aims: 1. To define further the binding of SV40 A protein (T antigen) to the origin of replication, 2. To construct a clone that isolates the minimal origin of replication from other viral regulatory elements, 3. To construct and characterize insertion, deletion, and base substitution mutations in regions of the origin not previously studied, 4. To define further the early replication events in vivo using wild-type and mutant origins, 5. To establish an in vitro system capable of initiating early events in DNA replication.